Global seroprevalence of scrub typhus: a systematic review and meta-analysis

Scrub typhus, a neglected disease, is a significant health concern in the Tsutsugamushi triangle of the Asia–Pacific and has raised global concerns due to recent cases occurring outside this region. To estimate the global prevalence of scrub typhus, we conducted a systematic review and meta-analysis. We conducted a systematic search of PubMed, Scopus, and Embase databases for observational studies on scrub typhus. Using a random-effects model, we combined the prevalence estimates with inverse-variance weights while also evaluating heterogeneity and publication bias. Among 3551 reports screened, we identified 181 studies with 1,48,251 samples for inclusion in our synthesis. The overall pooled seroprevalence (95% confidence intervals) of scrub typhus infections was 24.93% (23.27–26.60). Gender-wise pooled prevalence was estimated to be 50.23% (47.05–53.40) for males and 48.84% (45.87–51.80) for females. Eschar prevalence was observed to be 30.34% (22.54–38.15) among the positive cases. One-fourth of all the samples tested positive for scrub typhus and eschar was present in one-third of these total positive cases, encompassing regions beyond the Tsutsugamushi triangle. This estimation underlines the importance of this neglected disease as a public health problem. Strengthening surveillance and implementing disease control measures are needed in the affected regions.


Study participants
The participants of included studies were individuals positive for scrub typhus infection, based on Immunoglobulin M or G (IgM/ IgG) detection using Enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay (IFA), indirect immune peroxidase assay (IIPA), rapid diagnostic test (RDT), Weil-Felix test, or Polymerase chain reaction (PCR).

Data extraction and management
The two reviewers independently extracted information from the selected studies using a pre-designed data extraction form in Microsoft Excel.Extracted data included general information (author, study title, publication year, country), method information (study design, study setting, type of population, gender, study location, study period, laboratory investigations), and result information (number of Scrub typhus positives).Disagreements among the reviewers were resolved through discussion and consensus after consulting the third reviewer which has been elaborated in the Supplementary material S1 (page 50) 19 .The extracted data were analyzed using STATA software version 17 20 .

Risk of bias assessment
We used the AXIS tool 21 and Newcastle-Ottawa scale (NOS) 22 to assess the quality of analytical cross-sectional and cohort studies, respectively.The AXIS tool is designed to measure the aspects of study quality with 20 items, including justification of sample size, representativeness of the sample, a description of non-responders, use of validated measures, description of statistical methods, discussion of non-response bias and reporting of funding and conflicts of interest.The AXIS tool assesses the individual characteristics of a study cumulatively but not with a quantitative score.The risk of bias judgment was made through signaling questions and responses, namely "yes", "no", and "don't know".Quality evaluation through NOS was done by assigning up to nine items for the least risk of bias in three domains: (1) selection of study group (four items), (2) comparability of groups (two items) and (3) ascertainment of exposure and outcomes (three items) for cohort studies, respectively.Two authors independently assessed the risk of bias for each included study, and disagreements were discussed and resolved by consensus.

Statistical analysis
The pooled prevalence using the random effects (DerSimonian and Laird) method was reported as a proportion with a 95% confidence interval (CI).I 2 test was used to assess the evidence of heterogeneity.I 2 value < 25% was considered mild, 25-75% moderate, and > 75% was regarded as having substantial heterogeneity 23 .We used a Forest plot to represent the study-specific and pooled estimates for overall and subgroup analysis.A funnel plot was constructed to examine the publication bias (Supplementary Fig. S1).The subgroup analysis was conducted, and the pooled prevalence of Scrub typhus infections was estimated according to the study populations, study types, study settings (hospital vs. community; seasonal vs. non-seasonal), study areas, recent infections and past exposures.

Ethical approval
The study does not require ethical approval as it involves meta-analysis of publicly available research and utilized anonymized original data.

Characteristics of the studies
The characteristics of the included 181 studies are summarized in Supplementary Sero-positivity rates for different age groups were calculated and tabulated in Supplementary Table S3.Scrub typhus with coinfections was reported in fifty-six studies (Supplementary Table S4).The studies used a wide range of serological (non-specific and specific) and molecular techniques viz, Weil-Felix/ELISA/RDT/PCR/IFA/IIPA for scrub typhus detection (Supplementary Table S5).Twenty-five studies reported the type of O. tsutsugamushi strains.(Supplementary Table S6).

Risk of bias assessment
The quality of the studies assessed in this review ranged from low to moderate, with all 170 quantitative studies not meeting 8 out of 20 possible criteria of the AXIS tool.Of the studies that missed eight items, most did not take measures to address the concerns of non-response bias and outcome variables measured in accordance with the aims of the study.One hundred and fifty-nine studies (93.5%) lacked sample size estimation.One hundred and sixty-nine studies (99.4%) used a sample frame from a population closely representing the target or reference population.All the studies satisfied 12 of the 20 major criteria as per the AXIS tool (Supplementary Table S7).Newcastle-Ottawa quality assessment of cohorts rated most of the included studies to be good (Supplementary Table S8).

Meta-analysis
Based on the 181 studies, the pooled prevalence of scrub typhus was estimated to be 24.93%(23.27-26.60)(Fig. 2) which is around one-fourth of the population included in our meta-analysis.We found substantial heterogeneity between the studies that reported the outcome (I 2 = 99.45%).Based on gender, the pooled prevalence of scrub typhus among males was 50.23% (47.05-53.40),whereas, among females, it was 48.84% (45.87-51.80)(Supplementary Figs.S2, S3).
Studies were grouped based on the exposure status of the study population, i.e., currently infected (IgM) and previously exposed (IgG) populations.

Discussion
This systematic review and meta-analysis aims to provide a comprehensive quantitative assessment of the global prevalence of Scrub typhus infections.This meta-analysis incorporates diverse populations, such as blood donors, healthy volunteers, households, children, travelers, and pregnant women, affected by scrub typhus.The results of our analysis reveal a pooled prevalence rate of 24.93%, indicating that roughly one-fourth of all febrile cases worldwide are positive for scrub typhus.Moreover, our examination of previously exposed populations yielded an overall seroprevalence of 57.86%, suggesting that approximately half of the individuals tested had prior exposure to O. tsutsugamushi.The majority of cohort studies analyzed in our study reported positivity using IgM, indicating prevalence.However, four cohort studies reported scrub typhus based on IgG, potentially leading to an underestimation of prevalence.Furthermore, given that only three cohort studies reported scrub typhus based on IgG, the impact on the pooled prevalence is likely to be minimal.A variability in country-wise seroprevalence among the currently infected population was evident, with Iraq exhibiting notably high seroprevalence.This finding was based upon a rural community which had consistent interaction with animal reservoirs.Intriguingly, our analysis did not discern any significant gender predilection, as infection rates stood comparable between males and females.Furthermore, our estimations indicate that approximately 30% of individuals diagnosed with scrub typhus exhibit the presence of eschars, with variations observed across different countries highlighted in the Supplementary Fig. S4.The variations seen within countries and regions are directly related to how methodical and diligent the physicians were in examining their patients.Additionally, our analysis estimated a quarter of the tested population across different settings, including hospitals and communities, as well as in various study areas, encompassing urban, rural, and urban-rural regions to be exposed to scrub typhus infection.
The majority of studies in our analysis come from endemic (Tsutsugamushi triangle) and regions which earlier had not reported scrub typhus.Globalization and increased travel have expanded the reach of scrub typhus into areas earlier not known for reporting scrub typhus 16,24 , often presenting with nonspecific clinical symptoms.Limited awareness and diagnostic resources likely contribute to significant underreporting of scrub typhus cases, potentially explaining lower seroprevalence in island nations compared to others.Eschar presence varies widely (7% to 97%) among scrub typhus patients and the absence of eschar is reported to be an independent predictive factor for fatal outcomes 25 .Variability in eschar occurrence is noted across different O. tsutsugamushi strains 10,26 and among different ethnic populations 27,28 .Community-based studies are crucial for assessing disease exposure risk.Workplace and socioeconomic status are significant risk factors for human scrub typhus.Agricultural activities pose the primary risk for both rural and urban populations [Supplementary reference S1: 58].Deforestation and development projects lead to secondary growth of scrub vegetation, increasing mite and rodent populations [Supplementary reference S1: 28, 52, 112].Risk factors include housing type, rodents in peri-domestic areas, and forest interactions, with higher prevalence in rural areas [Supplementary reference S1: 107].Scrub typhus which was once thought to be a rural illness, is being increasingly recognized and diagnosed in urban settings [Supplementary reference S1: 75,108].The large population could have a significant effect on urban scrub typhus.Large urban populations, with their dense human presence, poor sanitation, and encroachment into natural habitats, coupled with environmental changes and increased man vector contact, create ideal conditions for the spread of scrub typhus.Urban scrub typhus should be regarded as one of the differential targets for febrile illness in areas where it is endemic.Urban environments differ from their rural surroundings.The eco-friendly trend of creating more green spaces such as parks in urban areas provides a conducive environment for rodent infestation resulting in such spaces acting as potential sites for scrub typhus transmission 29 .
Our review had some limitations that should be considered when interpreting the results.The high degree of heterogeneity observed among the included studies could have impacted the generated estimates.This heterogeneity could possibly be attributed to various factors, such as variations in the ethnicity of the study populations and differences in study methodologies, particularly design, sampling and diagnostic methods.Utilization of four cohort studies to report scrub typhus based on IgG may have contributed to an underestimation of the pooled prevalence.Few studies were included under different sub-groups, which might have overestimated their seroprevalence.Studies reporting equal numerator and denominator values were also excluded from the meta-analysis due to considerations of selection bias.Grey literature comprising academic papers, reports, dissertations, ongoing yet unpublished research as well as preprints were not included in our review.The exclusion of non-English publications might have resulted in a language selection bias in the study.

Conclusion
This systematic review and meta-analysis of the prevalence of scrub typhus highlights the worldwide spread of the disease.One-fourth of the population included in our meta-analysis were found to be affected by scrub typhus, with eschar being detected in about one-third of the scrub typhus confirmed cases.Males and females are at equal risk of getting infected.These findings extend to regions beyond the Tsutsugamushi triangle.This expansion beyond the endemic areas emphasizes the need for enhanced surveillance involving those regions.Monitoring disease trends and deploying public health interventions targeting vector and rodent control in the endemic areas is needed to tackle O. tsutsugamushi transmission to reduce incident cases.